Immune cells such as macrophages and activated lymphocytes are highly sensitive to osmotic stress from exposure to hyperosmotic environments (such as lymphoid tissues and local inflammation) or active cell proliferation. Cells adapt to osmotic stress by accumulating compatible osmolytes, which allow cells to efficiently retain intracellular water, maintain cytoplasmic volume, and dilute cellular ions and other critical solutes. Accumulating osmolytes significantly raise the intracellular solution osmolality, or equivalently reduce water activity. Macromolecular interactions coupled to large changes in hydration are therefore sensitive to osmotic changes inside the cell. PU.1 is an ETS-family transcription factor that regulates the development and function of immune cells such as macrophages and lymphocytes. Our recent studies have demonstrated that DNA-binding affinity and sequence selectivity for PU.1 are profoundly sensitive to their osmotic environment, but not for its close structural homolog, Ets-1. Independently, genomic studies show that regulation of PU.1-target genes, but not Ets-1 target genes, depend strongly on a functional osmotic stress response. Based on this evidence, we hypothesize that osmotic sensitivity is a mechanism for biological responsiveness and target specificity for PU.1. To test our hypothesis, we will determine the physicochemical nature of osmotic sensitivity by PU.1 and identify the structural elements that confer its osmotic sensitivity. We will use Ets-1 as an osmotically insensitive standard to interpret how osmotic sensitivity is specifically incorporated into PU.1, and how osmotic sensitivity affects site selectivity (the primary determinant of gene-activating potential) These proposed studies complement more than two decades of cellular and functional studies of PU.1 and other ETS transcription factors in culture and in vivo. They have specific relevance to how PU.1 and Ets-1 coordinately modulate their activities to permit correct maturation of T lymphocytes in the hyperosmotic thymus environment. Upon completion of this research, we expect to have established osmotic sensitivity as a mechanism for responsiveness by PU.1 to the cellular osmotic stress response program. More broadly, this research brings attention to hydration as a biophysical basis for responsiveness to physiologic osmotic stress in human cells. This knowledge has potential implication for our understanding of anisotonic disorders (such as diabetes mellitus and inflammation) associated with pathological intracellular osmotic stress.